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PubMed1976

Inhibition of Neisseria gonorrhoeae by a bacteriocin from Pseudomonas aeruginosa.

Supernatants from broth-grown cultures of Pseudomonas aeruginosa PA 103 exhibited bactericidal activity against Neisseria gonorrhoeae. The concentration of the bactericidal substance increased significantly after induction by mitomycin C. Purification was effected by salt fractionation, chromatography on diethylaminoethyl-cellulose, and sedimentation by centrifugation at 100,000 x g for 90 min. Electron microscopy of this purified preparation revealed structures resembling R-type pyocins in both the contracted and uncontracted state. Pyocins in the contracted state were observed in association with the gonococcal cell surface. No loss of bactericidal activity was observed after treatment with proteolytic enzymes. Standard pyocin typing procedures identified the pyocin pattern as 611 131. The bactericidal activity of this pyocin was examined on various species of Neisseria. Out of 56 strains of N. gonorrhoeae from disseminated and nondisseminated infections, all were susceptible to pyocin 611 131. However, only 3 of 20 strains of N. meningitidis and 5 of 16 strains of N. lactamica were susceptible. The bactericidal activity that pyocin 611 131 has for N. gonorrhoeae and other species of Neisseria is significant because it departs from the expected specificity that heretofore has distinguished bacteriocins from most "classical" antibiotics.

Bacteriocins↗
PubMed1982

A simple photometric method for determination of the activity of pyrocin R1.

A simple photometric method for rapid and accurate determination of the activity of pyocin R1, a bacteriocin produced by Pseudomonas aeruginosa strain P15, has been developed. This method is based on the turbidity-decrease observed when the bacteriocin is added to a suspension of sensitive bacteria P. aeruginosa strain P11. Optimum conditions for the turbidity-decreasing activity of pyocin R1 are in 0.01 M Tris-HCl buffer containing 0.2 M NaCl (pH 7.5) at 37 degrees C. A good correlation was found between the dose of pyocin R1 and the rate of the turbidity-decrease (with a correlation coefficient of more than 0.98). The amount of pyocin R1 required for this assay is nearly the same as that used for the conventional colony-counts method. The assay for one sample takes less than 3 min, whereas an overnight wait is necessary for the conventional method. This method is shown to be very suitable for following the time course of activity change observed when pyocin R1 is treated with various chemicals, including receptor substances obtained from sensitive cells. The turbidity-decrease assay was also found to be applicable to the determination of activities of other R-type pyocins.

Bacteriocins↗
PubMed1980

Effect of environment on sensitivity of Neisseria gonorrhoeae to Pseudomonas aeruginosa bacteriocins.

The effect of environmental variation on the susceptibility of Neisseria gonorrhoeae to pyocin produced by Pseudomonas aeruginosa was examined. Susceptibility to at least one pyocin was demonstrated in strains of N. gonorrhoeae (99%), N. meningitidis (35%), and N. lactamica (47%). The degree of sensitivity to pyocin displayed by N. gonorrhoeae was affected by varying the pH of the growth environment. Gonococcal strains were more sensitive to growth inhibition by pyocins at an alkaline pH and less sensitive to growth inhibition at an acid pH. Inhibitory titers fluctuated during nonselective subculture of fresh clinical isolates. There was no apparent correlation between auxotype and sensitivity to pyocin. Also, no relationship between colony morphology and pyocin sensitivity was seen.

Bacteriocins↗
PubMed1964

"CELL-WALL PROTEIN A" OF PSEUDOMONAS AERUGINOSA AND ITS RELATIONSHIP TO "ORIGINAL ENDOTOXIN PROTEIN".

Homma, J. Yuzuru (University of Tokyo, Tokyo, Japan), and Nachiko Suzuki. "Cell-wall protein A" of Pseudomonas aeruginosa and its relationship to "original endotoxin protein." J. Bacteriol. 87:630-640. 1964.-To compare the properties of two kinds of proteins, one obtained from the cell wall of Pseudomonas aeruginosa and the other from the endotoxin in the autolysate, the proteins were purified to such an extent that they were proved to be homogeneous by zone electrophoretic and ultracentrifugal analyses. Although there were some differences between the two in such values as nitrogen and phosphorus contents and sedimentation constants, their protein portions were found similar to each other in the ratio of amino acid compositions. It was proved serologically that the two proteins possessed a common specific antigen. They were found to be the same in their potencies in eliciting the Shwartzman phenomenon and pyrogenic reaction. The results of pyocine tests with various sensitive strains revealed that, against the sensitive strains, the spectra of both proteins were almost the same. Their pyocine activities were destroyed through digestion by protease, trypsin, and Nagarse. Either of the rabbit antisera against the two proteins could neutralize pyocine activities of both proteins. Serum-absorption tests proved that the pyocine-neutralizing antibodies of both antisera could be completely absorbed with each of the proteins. Under appropriate conditions, their pyocine activities were masked partially in vitro by the lipopolysaccharde of the endotoxin.

Amino Acids↗
PubMed1988

Marked increase of Pseudomonas aeruginosa serotype 012 in Belgium since 1982.

Routine typing was performed on a total of 7089 Pseudomonas aeruginosa strains isolated in 16 Belgian hospitals in the period from 1977 to 1986. The annual number of strains received ranged from 318 to 1346. The incidence of serotype O:12 was less than 2% until 1981 when it rose to 4%, steadily increasing to become the predominant serotype in 1984 (22%), 1985 (18%) and 1986 (22%). Since 1980 the O:12 isolates have exhibited characteristic patterns on pyocin and phage typing, 89% of O:12 isolates belonging to pyocin types 1, 39, 43, 45 or 105, whereas only 51% of isolates of other serotypes belonged to those pyocin types. Ninety-three per cent of serotype O:12 isolates belonged to phage types 68/119x, 68 or 119x, or were non-typable, whereas only 24.37% of other serotypes isolates exhibited these phage patterns. These distinctive patterns of pyocin and phage types suggest a high degree of homogeneity within the O:12 strains isolated in recent years in Belgium. Multi-centre or country-wide survey of Pseudomonas aeruginosa strains isolated in hospitals using epidemiological markers may be of value in identifying a sudden increase in epidemic strains.

Anti-Bacterial Agents↗
PubMed2000

Epidemiologic characterization of Pseudomonas aeruginosa in patients with cystic fibrosis.

OBJECTIVE: To determine persistence and variability of colonization with Pseudomonas aeruginosa in cystic fibrosis patients over long time periods, and to look for possible cross-colonization. METHODS: In total, 469 Pseudomonas aeruginosa isolates were obtained from 30 patients during the period from April 1994 to April 1996. The sources were mainly sputum and a few deep throat swabs. All grown strains dissimilar in macromorphology were processed separately. Typing with PFGE was carried out by contour-clamped homogeneous electric field electrophoresis. Genomic DNA was subjected to the rare-cutting restriction enzyme SpeI. For pyocin typing, the procedure described by Fyfe was applied. RESULTS: After typing with PFGE, we observed 40 restriction profiles. Eighteen different pyocin types were found. The most frequent pyocin type was type 3, followed by types 1 and 5. Twenty-two patients were persistently colonized by one clone specific and different for each patient, and four were co-colonized by a second clone also different for each of these patients. Cross-colonization had apparently been rare in the cystic fibrosis center of Leipzig. CONCLUSIONS: Typing with PFGE is well suited for detailed investigations of colonization with Pseudomonas aeruginosa in cystic fibrosis patients. Pyocin typing can provide additional information for epidemiologic purposes.

Adolescent↗
PubMed2005

Ciprofloxacin induction of a susceptibility determinant in Pseudomonas aeruginosa.

With few novel antimicrobials in development, resistance to the current selection of antibiotics increasingly encroaches on our ability to control microbial infections. One limitation in our understanding of the basis of the constraints on current therapies is our poor understanding of antibiotic interactions with bacteria on a global scale. Custom DNA microarrays were used to characterize the response of Pseudomonas aeruginosa to ciprofloxacin, a fluoroquinolone commonly used in therapy against chronic infections by this intrinsically resistant bacterium. Of the approximately 5,300 open reading frames (ORFs) on the array, 941 genes showed statistically significant (P </= 0.05) differential expression in response to 0.3x MIC of ciprofloxacin; 554 were promoted and 387 were repressed. Most striking among the responsive genes was the region between PA0613 and PA0648, which codes for the bacteriophage-like R2/F2 pyocins. In this region, virtually every ORF was increased by 0.3x MIC of ciprofloxacin and even more dramatically up-regulated (7- to 19-fold) following treatment with 1x MIC of ciprofloxacin. Pyocin gene expression was confirmed with lux reporter mutants and real-time PCR studies; pyocin-like particles were also present in transmission electron micrographs of supernatants from cells treated with 1x MIC of ciprofloxacin. Interestingly, mutants in this region exhibited >/=8-fold-increased resistance to ciprofloxacin and other fluoroquinolones, demonstrating that this region is a susceptibility determinant. Since this region is known to be variably present in the genomes of clinical isolates of P. aeruginosa (R. K. Ernst et al., Environ. Microbiol. 5:1341-1349, 2003, and M. C. Wolfgang et al., Proc. Natl. Acad. Sci. USA 100:8484-8489, 2003), these findings demonstrate that the R2/F2 pyocin region is a "loaded gun" that can mediate fluoroquinolone susceptibility in P. aeruginosa.

Anti-Infective Agents↗
PubMed1988

Selection and immunochemical analysis of lipooligosaccharide mutants of Neisseria gonorrhoeae.

The identification of enterobacterial mutants that contain alterations in the lipopolysaccharide (LPS) oligosaccharide core structure facilitated the development of the model of the physicochemical and immunochemical structures of enteric LPS. Results of recent immunochemical studies have suggested that the structural model of the lipooligosaccharides (LOSs) of Neisseria gonorrhoeae may differ from the enteric LPS model. The difficulties in the analysis of the wild-type gonococcal LOS have precluded understanding of the precise nature of the LOS structure. This study was undertaken to isolate a series of mutants of N. gonorrhoeae 1291 that had sequential saccharide deletions in the LOS. Results of preliminary studies suggested that the pyocin, designated pyocin C, allowed selection of gonococci with such mutant LOS structures. Results also indicated that the receptor for pyocin C binding was an LOS component. Pyocin C selection led to the isolation of five strains with LOS patterns on sodium dodecyl sulfate-polyacrylamide gels which differed from the LOS of parent strain 1291. In this system, the Mr of the parent LOS was 4,715, while the LOSs from the mutant strains demonstrated progressive saccharide deletions, with Mrs of 4,230, 4,089, 3,627, 3,262, and 3,197. Protein patterns of these mutants on sodium dodecyl sulfate-polyacrylamide gels were qualitatively similar to those of the parent strains. Results of studies with five monoclonal antibodies specific for neisserial LOS indicated that shared as well as unique epitopes were present on the mutant LOSs. Results of ketodeoxyoctonate analysis of the mutant LOSs indicated that the majority of the ketodeoxyoctonate residues may be substituted on C-4 or C-5. Chemical and immunological analysis of such LOS mutants should expedite the development of the model for the structure of gonococcal LOS.

Antibodies, Monoclonal↗
PubMed2006

Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1.

The purpose of this study was to clone the carocin S1 gene and express it in a non-carocin-producing strain of Erwinia carotovora. A mutant, TH22-10, which produced a high-molecular-weight bacteriocin but not a low-molecular-weight bacteriocin, was obtained by Tn5 insertional mutagenesis using H-rif-8-2 (a spontaneous rifampin-resistant mutant of Erwinia carotovora subsp. carotovora 89-H-4). Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of the contiguous 2,280-bp region were determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequence fragment. ORF2 and ORF3 were identified with the carocin S1 genes, caroS1K (ORF2) and caroS1I (ORF3), which, respectively, encode a killing protein (CaroS1K) and an immunity protein (CaroS1I). These genes were homologous to the pyocin S3 gene and the pyocin AP41 gene. Carocin S1 was expressed in E. carotovora subsp. carotovora Ea1068 and replicated in TH22-10 but could not be expressed in Escherichia coli (JM101) because a consensus sequence resembling an SOS box was absent. A putative sequence similar to the consensus sequence for the E. coli cyclic AMP receptor protein binding site (-312 bp) was found upstream of the start codon. Production of this bacteriocin was also induced by glucose and lactose. The homology search results indicated that the carocin S1 gene (between bp 1078 and bp 1704) was homologous to the pyocin S3 and pyocin AP41 genes in Pseudomonas aeruginosa. These genes encode proteins with nuclease activity (domain 4). This study found that carocin S1 also has nuclease activity.

Amino Acid Sequence↗
PubMed1999

Epidemiological analysis of Pseudomonas aeruginosa strains isolated from a selected patient population in Brno, Czech Republic.

In the 1996/97 period, 1,413 Pseudomonas aeruginosa (PA) strains were isolated from 843 patients of the Brno teaching hospitals of St. Anne and Bohunice together with small groups from other hospitals. In the same period, 203 PA strains, used as controls, were isolated from 187 patients treated outside hospitals. Statistical evaluation was based on 1,023 hospital isolates and 189 control strains. A total of 16 isolates were recovered from the hospital environments and two from therapeutic swimming pools. The epidemiological analysis of these PA strains was based on pyocin typing, serological typing and phage typing. The most frequently occurring pyocin types amongst our strains fell into 8 pyocin-type groups. The prevailing groups differed significantly between the hospital patient and control groups. Similarly, serological typing identified differences in the predominant serotypes between hospital and control patients. The phage typing method revealed that the control PA strains were significantly more sensitive to 21 polyvalent bacteriophages used than the hospital isolates. In relation to pyocin and serological typing, strains isolated from the hospital environment showed characteristics similar to those of the PA strains isolated from hospital patients. Our results indicate that the majority of strain isolated from hospitalised patients had their origin from human or inanimate contacts in the hospitals.

Bacteriophage Typing↗
PubMed1990

Typing of polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients.

The study assesses the reproducibility, typability and discriminatory power of several typing methods for Pseudomonas aeruginosa isolated from cystic fibrosis patients. 178 polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients were serotyped using polyclonal sera and monoclonal antibodies, phage typed, pyocin typed and reverse phage typed. 31 of these polyagglutinable isolates, six monoagglutinable isolates and three nontypable isolates were also typed by means of hybridization using a DNA probe. In a comparison of the methods used, on polyagglutinable isolates only, typability was 0% with polyclonal sera, 90% with monoclonal sera, 94% with phage typing, 85% with pyocin typing, 36% with reverse phage typing and 100% with DNA-prope typing. Using monoclonal antibodies, the reproducibility was 75%, while that of phage typing was 88%, pyocin typing 53% and reverse phage typing 62%. Typing with the DNA probe was not repeated. using polyclonal sera, repeated typing showed that 94% of the isolates were polyagglutinable. Using phage typing, 40% of the isolates belonged to phage type 31, while 60% were distributed amongst 32 phage types. Using monoclonal antibodies, 71% of the isolates belonged to 0-group 3, and these isolates showed 16 different phage types. Subdivision of the phage types was further achieved by both pyocin typing and reverse phage typing. The DNA probe typing made it possible in some cases to discriminate between isolates which were otherwise found identical with the conventional typing methods, while in other cases typing with the DNA probe recorded as identical isolates which conventional methods had typed as being different. These differences may be due to a high mutation rate caused by the selection pressure of antibiotics, and by the host immune response. According to our results, investigations of reproducibility and typability of old and new typing methods are essential when they are used in clinical situations. The low reproducibility of some of the typing methods in the present study affects the reliability of epidemiological investigations in cystic fibrosis patients. Usage of only one method may not be sufficient in cases of polyagglutinable strains from cystic fibrosis patients.

Agglutination Tests↗
PubMedDate not supplied

Endogenous and exogenous infection with Pseudomonas aeruginosa in a burns unit.

Twenty patients who were admitted to the Burns Unit from December, 1969 through October, 1970 were studies to determine the sources of infection caused by Pseudomonas aeruginosa. The pyocine typing method was employed for finger printing of 383 isolates recovered from wounds and 67 isolates from environmental cultures of nurses' hands, sinks, floors, bed rails, walls and baths. In addition, cultures of moist rectal swabs were carried out daily for the first six days of hospitalization to assess the importance of endogenous infection. In six patients, the rectum was identified as the source of infection. However, in these patients, pyocine types of Ps. aeruginosa which were not obtained from rectal cultures, were also recovered. Pyocine types 1b, 10 and 31 were isolated more frequently than others. Clustering of common pyocine types suggests cross-contamination. Sinks were found to be consistently contaminated with Ps. aeruginosa. Amongst the environmental sources, positive cultures were occasionally obtained from floors, bed rails and nurses' hands. It is suggested that sinks are probably the most important reservoir of Pseudomonas infection in burns.

Burn Units↗